Project Summary Clostridium perfringens type F strains are the 2nd most common cause of bacterial food poisoning (FP) in the USA, where ~1 million cases/year occur. These bacteria also cause many cases of nonfoodborne hu- man intestinal diseases (NFD), such as antibiotic-associated diarrhea. The virulence of type F strains re- quires production of Clostridium perfringens enterotoxin (CPE). All type F diseases are true infections where type F strains initially multiply in the intestines but then produce CPE when they sporulate in vivo; this toxin is released into the intestinal lumen upon lysis of the mother cell to free its mature spore. Type F infections are typically a diarrheal disease but can also involve lethal enterotoxemia (where CPE produced in the intestines is absorbed to damage internal organs like the liver) in patients with certain predisposing medical conditions. In addition to CPE, all type F NFD strains and ~50% of type F FP strains produce a secreted sialidase named NanI. For those type F strains, NanI is their predominant exosialidase and this sialidase is produced in both vegetative cultures and sporulating cultures (where NanI is co-present with CPE). NanI is emerging as an important virulence factor for NanI+ type F strains, e.g., we showed NanI sialidase contributes in vitro to growth, sporulation and CPE production and to persistent intestinal colonization. We also reported that, i) NanI enhances CPE binding/cytotoxicity for Caco-2 cells and ii) contact with small intestinal fluid, as occurs during type F enteric infections, proteolytically processes NanI to a 60 kDa fragment that possesses increased sialidase activity and greater ability than native NanI to promote CPE binding/cytotoxicity for enterocyte-like Caco-2 cells. While producing cell surface sialyl-conjugates, Caco-2 cells make minimal mucus, which is heavily sialylated and abundant in the intestines. Therefore, we hypothesize, i) NanI is even more impactful for promoting CPE activity in the presence of substantial mucus, as occurs in the intestines, ii) this effect is enhanced by proteolytic activation of NanI by intestinal proteases, and iii) NanI promotes type F infection/diseases. This project will test those important hypotheses using in vitro and in vivo models of CPE activity or type F infection/diseases. Aim 1 will employ enterocyte-like cell culture models that do or do not produce substantial mucus to compare the relative impact of NanI or proteolytically-activated NanI on promoting CPE cytotoxicity or CPE paracellular transit (an in vitro surrogate for CPE absorption from the intestines during enterotoxemia) in the absence vs. presence of mucus. Aim 2 will use animal infection models, i.e., rabbit and mouse small intestinal loop models of enteritis or enterotoxemia, respectively, to test if NanI contributes to type F infection/diseases and characterize NanI effects that could contribute to virulence, i.e., does NanI increase CPE activity/transit and/or type F strain growth, sporulation or CPE production in the intestines? If NanI is shown to potentiate type F infections, it would suggest a future translational approach, i.e., using sialidase inhibitors, to ameliorate these diseases.